Attachment II - Page 3 
Table II. Toxicity Data 
!• Estimate of parenteral LD^q of purified Shiga or Shiga-like toxin for 
monkeys . 
- The parenteral LD^q purified Shiga or Shiga-like toxin for a 
20 gram mouse is approximately 1 pg (O'Brien et al. )« 
- The lethal dose of crude Shiga toxin for a parenterally-challenged 
20 g mouse and a 5 lb monkey is equivalent (Branham et al. 1953). 
- More than 50% of total toxin activity in crude extracts is recovered 
after purification. Therefore, Shiga and Shiga-like toxin retain 
most or all of their toxicity during purification. 
Based on these observations, the parenteral LD^q for monkey of pure 
Shiga or Shiga-like toxin is estimated to be approximately 1 yg. 
II. Estimate of toxicity of Shiga toxin instilled into monkey intestinal 
pouch. 
- The maximum number of mouse parenteral LD^q instilled into the monkey 
intestinal pouch was 12,000. No adverse effects were observed (see 
Branham _et_ _al^. , 1953). 
- The amount of pure toxin equivalent to 12,000 mouse purified LD^q is 
approximately 12,000 yg. Therefore, 12 mg of pure toxin is estimated 
to be below the threshold for toxicity when given intraintestinally 
In monkeys . 
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III. Estimate of maximum amount of Shiga-like toxin produced by 2 x 10 cells 
of _E_. coll . 
- Five mis of a 4 x 10^/ml overnight culture of _E. coll 0157 :H7 
(representative of the most highly toxinogenic clinical isolates) 
contained 2 x 10 bacteria and a total of 1 x 10 50% cytotoxic doses 
(CD^q) of Shiga-like toxin. Approximately 10% of the toxin was found 
in the culture supernatant, and the remaining 90% was recovered in 
sonic extracts of the bacteria. 
- This represents the highest toxin yield for bacteria grown under 
optimal conditions in vitro . In media that are not optimal for toxin 
production, the yield of toxin often decreases by at least 10-100 
fold . 
- One yg of purified Shiga toxin corresponds to 10^ CD^q. 
The maximum yield of Shiga-like toxin from 2 x 10^ _E _. coli 0157 :H7 
corresponds to approximately 1 yg of pure toxin. 
- If strains constructed by recombinant DNA technology could produce x- 
fold higher yields of Shiga-like toxin per cell, the maximum yield 
per 2 x 10 ° cells would increase by the factor x. 
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